Isolation, purification and characterisation of lunasin from defatted soybean flour and in vitro evaluation of its anti-inflammatory activity

Lunasin is a chemopreventive peptide present in soybean and other plant sources. The high cost involved in obtaining synthetic lunasin limits its application in chemopreventive and nutritional interventions. The objective of this study was to isolate, purify and characterise lunasin from defatted soybean flour and determine its in vitro anti-inflammatory activity using RAW 264.7 macrophages. Isolation and purification was achieved by ion-exchange chromatography, ultrafiltration and size exclusion chromatography. The identity of lunasin was established by Western blot, HPLC, MALDI-TOF and LC/MS-MS. The results showed that lunasin eluted from a DEAE anion exchange column at 0.15 M NaCl, and after 1.5 void volumes from size exclusion chromatography. Fractions from both chromatographic techniques consistently showed three peptides with positive immunoreactivity against lunasin mouse monoclonal antibody corresponding to 5, 8 and 14 kDa. LC/MS-MS analysis of the three immunoreactive peptides showed that 5 and 14 kDa bands contained the lunasin epitope, RGDDDDDD DDD while 8 kDa band showed high homology with 2 S soy albumin, a lunasin precursor.This is the first report on the potential anti-inflammatory activity of lunasin. Treatment of RAW 264.7 macrophage with 100 μM lunasin decreased the production of NO (92.6 ± 0.8%) and PGE2 (10.1 ± 4.5%), and the expression of iNOS (27.8 ± 2.1%) and COX-2 (41.4 ± 16.7%). We concluded that combination of ion-exchange chromatography, ultrafiltration and size exclusion chromatography represents a feasible process for the production of purified lunasin, which was also found to inhibit COX-2/PGE2 and iNOS/NO pathways. This newly discovered property of lunasin might contribute to the suppression of inflammation in vivo.